biostudies-arrayexpressGizem KarsDrosophila melanogasterhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15796The DOM-A complex regulates cell growth and proliferation in Drosophila. Analogous to the mammalian Tip60–p400 complex, DOM-A integrates two epigenetic effectors: a SWR1-type ATPase (Domino-A) that mediates histone exchange and the Tip60/KAT5 acetyltransferase. We identified Xbp1, a conserved transcriptional regulator of the unfolded protein response (UPR), as a tight interactor of the immunopurified DOM-A complex and explored the functional consequences of this association. Integrative analysis of Xbp1 and DOM-A occupancy in proliferating cells, together with reciprocal protein depletion, revealed two distinct modes of Xbp1 chromatin binding. In a sequence-specific mode, Xbp1 recruits DOM-A to motif-bearing promoters of UPR genes. In a second, motif-independent mode, Xbp1 localizes to hundreds of high-confidence DOM-A binding sites that lack Xbp1 recognition motifs; these interactions depend on DOM-A, consistent with a “reverse targeting” mechanism. Consistent with functional coupling, depletion of DOM-A reduces Xbp1 protein abundance without affecting Xbp1 mRNA levels, suggesting post-translational stabilization of Xbp1 upon association with DOM-A. Together, these findings indicate that the genome-wide interplay between Xbp1 and DOM-A may integrate UPR signaling with the broader, DOM-mediated regulation of cell growth and proliferation.biostudies-arrayexpressSample Collection - CUT&RUN was performed as previously described (Meers M.P. et.al., 2019. DOI: 10.7554/eLife.46314). For each condition, 10⁶ cells were washed three times with Wash Buffer (WB: 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, cOmplete EDTA-free Protease inhibitor), centrifuging at 600 x g for 3 min. Cells were resuspended in 1 mL WB per antibody. Concanavalin A beads (10 μL/sample) were activated by two washes in Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), mixed with cell suspension and incubated for 10 min at RT with rotation. Beads were collected on a magnet and resuspended in 150 μL Antibody Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, 2 mM EDTA, 0.5 mM Na-butyrate) containing diluted antibodies, followed by incubation for 2 h at RT or overnight at 4°C with slow rotation. After two washes with Dig-Wash Buffer (DWB: 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.05% digitonin, cOmplete EDTA-free Protease inhibitor), beads were resuspended in 150 μL pAG-MNase solution (700 ng/mL in DWB) and incubated at 4°C for 1 h with slow rotation. After two further washes, beads were resuspended in 100 μL DWB, chilled, and digestion was initiated with 2 μL 100 mM CaCl2 for 30 min. Reactions were stopped with 100 μL 2x Stop Solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% digitonin, 100 μg/mL RNase A, 50 μg/mL glycogen) and incubated at 37°C for 30 min to release chromatin. Supernatants were collected on a magnet. Reverse crosslinking was done with 2 μL 10% SDS and 2.5 μL Proteinase K (20 mg/mL) at 50°C for 1 h. DREX-ChIP was performed in chromatinized genomic DNA. Briefly, Drosophila embryonic extract from the pre-blastoderm stage was incubated with DNA in the presence of ATP. Samples were mildly fixed with 0.1% formaldehyde for 10 min, quenched with 125 mM glycine for 5-10 min, and fragmented by 3 min MNase digestion to yield mononucleosomes. Reactions were adjusted to 1 mL with RIPA Buffer (25 mM HEPES-NaOH pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 0.1% sodium deoxycholate, freshly added 1 mM DTT, cOmplete EDTA-free Protease inhibitor). Protein A/G beads (20 μL slurry; Cytiva, Cat. Nos. 17-0618-05, 17-5280-05) were equilibrated in RIPA (3 x 5 min washes). For pre-clearing, samples were incubated with beads for 1 h at 4°C, then beads were removed. Antibody (1 μL) was added and incubated overnight at 4°C with rotation. The following day, 20 μL equilibrated beads were added for 3 h at 4°C, then washed three times with RIPA. DNA was eluted by incubating beads in 100 μL 1x TE with 3.2 μL 10% SDS and 10 μL Proteinase K (10 mg/mL) at 56°C overnight.Sequencing - Libraries were sequenced paired-end, 60x60bp with the NextSeq2000 platform.Sample Treatment - Double-stranded RNA (dsRNA) targeting the desired sequences was generated by in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit (NEB, Cat. No. E2040S). For CUT&RUN, the cells underwent two consecutive rounds of 6 μg dsRNA for a total of 7 days. Non-targeting dsRNA (GST) was included in the RNAi experiments as a negative control. For Xbp1 CUT&RUN cells were treated without or with 5 mM DTT for 1 hour prior to CUT&RUN.Growth Protocol - Cells were cultured at 26°C in Schneider’s Drosophila Medium (Thermo-Fischer, Cat. No. 21720024) supplemented with 10% FBS (PAN Biotech, Cat. No. P30-3031) and 1% Penicillin-Streptomycin solution (PenStrep; Sigma-Aldrich, Cat. No. P-4333).Nucleic Acid Extraction - DNA was purified by phenol-chloroform extraction, followed by ethanol precipitation (2 mg/mL glycogen, 500 μL 100% ethanol, 15 min on ice). Pellets were washed with 100% ethanol and resuspended in 30 μL 0.1x TE buffer.Library Construction - For CUT&RUN a modified version of NEBNext Ultra II DNA Library Prep Kit (NEB, Cat. No. E7645S) protocol was followed as described in (Kong et al., 2021. DOI: 10.1016/j.xpro.2021.100750 ). For ChIP, manufacturer`s protocol was carried out.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw reads from FASTQ files were first trimmed to remove sequencing adaptors using cutadapt. Trimmed reads were then aligned to the Drosophila melanogaster reference genome (Ensembl BDGP6.46) using Bowtie2. BigWig files were generated using bamCompare (deepTools). Each sample was normalized to the corresponding background control (input, IgG or PPI) and read depth.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 2000CUT&RUNDrosophila melanogasterGizem KarsPeter BeckerZivkos ApostoloufalseBinding site analysis and identification of reciprocal targeting of Dom-A and Xbp1 in Drosophila melanogasterThe DOM-A complex regulates cell growth and proliferation in Drosophila. Analogous to the mammalian Tip60–p400 complex, DOM-A integrates two epigenetic effectors: a SWR1-type ATPase (Domino-A) that mediates histone exchange and the Tip60/KAT5 acetyltransferase. We identified Xbp1, a conserved transcriptional regulator of the unfolded protein response (UPR), as a tight interactor of the immunopurified DOM-A complex and explored the functional consequences of this association. Integrative analysis of Xbp1 and DOM-A occupancy in proliferating cells, together with reciprocal protein depletion, revealed two distinct modes of Xbp1 chromatin binding. In a sequence-specific mode, Xbp1 recruits DOM-A to motif-bearing promoters of UPR genes. In a second, motif-independent mode, Xbp1 localizes to hundreds of high-confidence DOM-A binding sites that lack Xbp1 recognition motifs; these interactions depend on DOM-A, consistent with a “reverse targeting” mechanism. Consistent with functional coupling, depletion of DOM-A reduces Xbp1 protein abundance without affecting Xbp1 mRNA levels, suggesting post-translational stabilization of Xbp1 upon association with DOM-A. Together, these findings indicate that the genome-wide interplay between Xbp1 and DOM-A may integrate UPR signaling with the broader, DOM-mediated regulation of cell growth and proliferation.2025-10-20T00:00:00Z2026-05-27T12:42:34.669Z2025-10-20T13:43:05.962ZE-MTAB-15796ERP182516EFO_0002944EFO_0009973EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003969EFO_0004184